The TGF-beta family of ligands signal through a unique heteromeric receptor complex distinguished by its serine-threonine kinase activity. Recently, a direct signal transduction pathway from these receptors to nuclear target genes has been elucidated which involves a novel family of proteins termed Smads. In this pathway, receptor-activated Smads are phosphorylated directly by the type I receptor kinase and, in association with a common mediator Smad4, translocate to the nucleus where they participate in transcriptional complexes. We have taken a multi-faceted approach to gain insight into the biochemistry of this pathway in vitro and to understand its significance in vivo. One approach has been to identify unique factors which modulate receptor or Smad activity. Having previously identified a role for the TGF-beta-receptor-interacting protein, TRAP1, as a chaperone for the obligate signaling partner, Smad4, we are now characterizing a related protein which we call TRAP1-like protein, or TLP. TLP appears to have the unique feature of selectively blocking signaling from Smad3, but not Smad2. As such, it could play an important role in modulating the signal transduction pathways and gene targets of TGF-beta in specific cell types or in disease. These studies will be expanded to define the roles of these molecules further, to examine their specific patterns of cellular expression, and to conduct yeast two-hybrid screens to determine whether these proteins, in turn, interact with yet other components of the signaling complex. This approach has already potentially important interactors of SNIP1, another novel Smad-interacting protein which acts as a transcriptional repressor. Other studies are focused on identifying the unique functions of the Smad2 and Smad3 pathways in cells and tissues. We are addressing this by using microarray analysis to identify cDNAs uniquely induced by Smad3- or Smad2-dependent pathways in mouse embryo fibroblasts null for either Smad2 or Smad3. These studies have resulted in identification of targets of each of these signaling pathways, the characterization of which is providing a new perspective on immediate-early and second-order gene targets of TGF-beta. We are presently investigating the regulation of two of the Smad-dependent immediate-early genes which have been implicated in apoptosis and in epithelial-to-mesenchymal transition, respectively, Gadd45beta and Snail. To examine specific targets of Smad2 and Smad3 in cancer cells, we are characterizing the outcome of overexpression of Smad2 or Smad3 in tumorigenesis and metastasis by examining both in vitro and in vivo effects of altering the balance of these two pathways in human breast cancer cells derived from the parental MCF10A line. To complement the above basic biochemical approaches, we have also developed a strong program of research based on the hypothesis that deletion of specific downstream signaling pathways in vivo should, conceptually, have a less severe and more selective effect than broader-based approaches involving targeted deletion or overexpression of ligand or receptors. The Smad3 knockout mouse, developed by Chuxia Deng, NIDDK, is now providing new insights into the roles of TGF-beta in hematopoiesis, in repair of injury, and in fibrosis. In collaboration with Drs. Angelo Russo and James Mitchell, CCR, we are focusing on protective effects of loss of Smad3 in the skin in response to ionizing radiation, and attempting to correlate these findings with studies of effects of irradiation on primary keratinocytes and dermal fibroblasts in vitro. In a collaboration with Dr. Shizuya Saika, Wakayama U., we have shown that the pathologic transdifferentiation of lens epithelial cells to a mesenchymal phenotype post injury to the eye is completely blocked in mice lacking Smad3. Elucidation of pathogenetic mechanisms of TGF-beta dependent on the Smad3 pathway now suggest that development of a Smad3 inhibitor will have wide-ranging clinical applications in acceleration of epithelization of cutaneous wounds, in reducing opacification of lens implants, and in reducing fibrosis.